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In vitro rooting improvement of globe artichoke (cv. Camus de Bretagne) by GA3
SCIENTIA HORTlCULTuM ELSEVIER
Scientia Horticulturae 72 (1997) 59-62
Short communication
In vitro rooting improvement of globe artichoke (cv. Camus de Bretagne) by GA, J.M. Morzadec
*, A. Hourmant
Laboratoire de Physiologic des Syst&nes Intt?gr& Unicersite’ de Bretagne Occidentale, 6 ALjenue Victor le Gorgeu, B.P. 809, 29285 Brest Cedex, France
Accepted 25 July 1997
Abstract Microrosettes of globe artichoke (Cynara scolymus L.) were subcultured in a medium with naphthalene acetic acid and kinetin. For rooting, shoots were placed on an auxinic rooting medium (2.68 PM naphthalene acetic acid) supplemented with or without gibberellic acid (GA,). Treatments with 2.9 and 14.4 FM GA, resulted in 80.5 and 92.3% root formation, respectively, whereas 28.8 /IM GA, did not significantly change the rooting rate produced by auxin alone (50%). Moreover, the number of roots per shoot significantly increased for plants grown on rooting media containing 2.9 and 14.4 PM GA,. Stimulation by GA, of rhizogenesis was accompanied with a large increase of leaf growth. 0 1997 Elsevier Science B.V. Keywords: Cynara scolymus L.; In vitro rooting; Gibberellic acid (GA,)
1. Introduction
The
globe
propagated
artichoke
vegetatively
(Cynara by suckers
scolymus originating
L.) is a perennial from
axillary
buds
rosette
plant
on the basal
usually part of
et al., 1983; Basnizki and Goldschmidt, 1994). The efficiency of this method is however limited by the small proportion of axillary buds which produce viable cuttings (Benoit and Ducreux, 198 1). Besides, some heterogeneity is also introduced by the location of buds on mother plants producing differences in the the hypogeal
stem (Ryder
* Corresponding author. Tel.: +33 298 01 66 92; fax: +33 298 01 71 70; e-mail: jeammichel. [email protected] 0304.4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(97)00112-X
60
J.M. Morzadec, A. Houtmant/Scientia
Horticulturae 72 (1997) 59-62
inherent physiology of transplanted cuttings (Foury, 1976). In vitro vegetative multiplication techniques could, therefore, provide a way to correct these shortcomings. In vitro plant regeneration and propagation of globe artichoke has been studied for some forty years (Toponi, 1960). Microrosettes that develop have been found to be difficult to root, even in the presence of auxins. Depending on the cultivar, various results have been reported ranging from 1% rooted plants (Benoit and Ducreux, 1981) to 77% (Bigot and Foury, 1984) with 30% for cv. Camus de Bretagne (Moncousin, 1980). Because unrooted plants give rise to high mortalities during acclimatization, the present study was undertaken to improve rooting of cv. Camus de Bretagne. Gibberellins have been shown to inhibit rooting (Burkhart and Meyer, 1991); however, at low concentrations, they can stimulate it (Tizio et al., 1970). Gibberellins have long been known to play a major role in the organogenesis of rosette plants such as the globe artichoke (Basnizki and Goldschmidt, 1994). For these reasons, we have investigated the effects of the gibberellic acid (GA,) on rooting cv. Camus de Bretagne cultivated in vitro.
2. Materials
and methods
In vitro stock cultures of globe artichoke cv. Camus de Bretagne obtained from Organisation Bretonne de Selection (Plougoulm, France) were used as the source material. Cultures were subcultured for proliferation every 3 weeks on Murashige and Skoog (1962) basal medium supplemented with vitamins and Fe-EDTA (Linsmaier and Skoog, 1965), NaH,PO, (0.71 mM), adenine sulfate (0.22 mM), kinetin (2.3 PM), N6-(2-isopentenyl)adenine (2.46 PM) and naphtalene acetic acid (NAA) (0.54 PM) (PCcaut and Martin, 1993). For rooting, microrosette shoots selected presented at least a size of 5 cm and were cut 2 cm above their base before being transferred to a rooting medium. The Murashige and Skoog (1962) basal medium with l/2 strength for macroelements supplemented with vitamins and Fe-EDTA (Linsmaier and Skoog, 19651, 2 g 1-l charcoal activated, 2.68 PM NAA (medium A) was used and various concentrations of GA, 2.9, 14.4 and 28.8 PM were added. All media were supplemented with 30 g 1-l sucrose and 8 g 1-l or 7 g l- ’ agar type-E (Sigma Chemical, St. Louis, USA) for proliferation medium or rooting media, respectively. The pH of the complete media was adjusted to 5.8 with NaOH before autoclaving at 120°C for 20 min. Cultures were propagated in 15 X 2.2 cm tubes with 10 ml culture medium covered with clean plastic caps. Plants were grown at 23°C f 1, under 16 h day-’ light with an intensity of 100 PE me2 s-’ provided by Sylvania Grolux fluorescent lamps. Percentages of rooting were determined once a week during 6 weeks; the number of roots per shoot and analysis of fresh and dry weights were performed on rooted rosette shoots 6 weeks after subculturing. Results are the means of 4 independent experiments with 40 shoots per condition. Statistical analyses were performed using x2 tests and means separation using the Student’s t-test with a significance level of 5%.
J.M. Morzadec, A. Houmant/Scientia
Horticulturae
72 (1997) 59-62
61
3. Results and discussion Rosettes subcultured on rooting media (A, 2.9, 14.4 or 28.8 FM GA,) developed only one shoot apices per rosette. When globe artichoke rosettes raised in vitro on the multiplication medium were transferred to the rooting auxinic medium (A), a poor rooting of the plants was observed during the first three weeks; then the percentage of rooted explants started to increase, reaching 50% at week 6 (Fig. 1). The addition of GA, to the auxinic medium improved the rooting rate depending on GA, concentration. At the lowest concentrations (2.9 and 14.4 PM), GA, stimulated significantly (P = 0.05) the root formation after two weeks of culture. At 14.4 ,uM, GA, was significantly (P = 0.05) more effective than at 2.9 PM, producing at week 6 a high rooting percentage that was 92.3% (Fig. 1). The addition to the culture medium of a higher concentration of GA, (28.8 PM) did not change significantly (P = 0.05) the percentage of rooted explants compared to the auxinic medium (A); this was true through all the duration of culture (Fig. 1). The action of GA, on the root formation seems to be associated with an effect on the growth of microrosettes. Thus, at week 6, GA, (2.9 and 14.4 PM) significantly stimulated both the fresh and dry weights of rooted rosette shoots while 28.8 PM GA, did not have any effect (Table 1). The mean number of roots per shoot was statistically increased with 2.9 and 14.4 PM GA, whereas 28.8 PM GA, had no significant effect (Table 1). The data shows an unusual stimulating effect of GA, in rooting of a rosette plant; this phytohormone increased both the percentage of rooting (Fig. 1) and the number of roots per plant (Table 1). Because an earlier root appearance in GA,-treated shoots
Fig. 1. Effects of different concentrations
of GA, added to medium A on rooting of globe artichoke plants.
62
J.M. Morzadec, A. Hourmant/Scienria
Horticulturae 72 (1997) 59-62
Table 1 Effects of GA, added to the auxinic medium (A) on the fresh weight @WI, the dry weight (DW) of rooted rosette shoots and on the number of roots. Results expressed in g FW or DW ( f SE) and in number of roots per plant. Data recorded after 6 weeks of rooting induction. Means within a line followed by the same letter are not significantly different at P = 0.05 according to the Student’s t-test
Leaf FW Leaf DW Number of roots
Medium A
GA, (PM) 2.9
14.4
28.8
1.84*0. 13 a 0.14&O. 01 a 1.85+0. 15 a
2.71 +O. 17 b 0.19*0. 01 b 2.25&O. 13 b
3.41 +o. 15 c 0.23+0. 01 c 2.30*0. 09 b
1.79+0. 14 a 0.13*0. 01 a 1.95&O. 12 a
could facilitate the absorption of both nutrients and water, one could explain the improved growth of leaves recorded after 6 weeks of culture (Table 1). The use of GA, in the rooting medium conducts to a rapid root expression and to explants of high quality. Additional studies could be realised with other cultivars of globe artichoke especially cv. Violet de Provence known to be difficult to root.
References Basnizki, .I., Goldschmidt, E.E., 1994. Further examination of gibberellin A, effects on flowering of globe artichokes (Cynara scolymus L.) under controlled environment and field conditions. Isr. J. Plant Sci. 42, 159-166. Benoit, H., Ducreux, G., 1981. Etude de quelques aspects de la multiplication vCg&ative in vitro de l’artichaut (Cynara scolymus L.). Agronomie 1, 225-230. Bigot, C., Foury, C., 1984. Multiplication in vitro d’artichaut (Cynara scolymus L.) ZI partir de semences: Comparaison au champ de quelques clones ?I la lignte dont ils sont issus. Agronomie 4, 699-710. Burkhart, L.F., Meyer, M.M. Jr., 1991. The gibberellin synthesis inhibitors, ancymidol and flurprimidol, promote in vitro rooting of white pine microshoots. Plant Cell Rep. 10, 475-476. Foury, C., 1976. L’Artichaut Bul. Tech. Informa. 311, 415-432. Linsmaier, E.M., Skoog, F., 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18, 100-127. Moncousin, C., 1980. Multiplication vtgCtative acc&rie de Cynara scolymus L. I. RCsultats prkliminaires. Rev. Hart. Suisse 53, 149-154. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497. PBcaut, P., Martin, F., 1993. Variation occurring after natural and in vitro multiplication of early Mediterranean cultivars of globe artichoke (Cynara scolymus L.). Agronomie 13, 909-919. Ryder, E.J., de Vos, N.E., Bari, M.A., 1983. The globe artichoke (Cynara scolymus L.). HortScience 18, 646-653. Tizio, R., Saussay, R., Gautheret, R., 1970. Action de quelques gibtirellines sur la rhizogenese de fragments de rhizomes de Topinambour cultivts in vitro. C. R. Acad. Sci. Paris, S&e D 270, 2088-2092. Toponi, M., 1960. Recherches sur le d&eloppement de fragments de bracdes d’artichaut. C. R. Acad. Sci. Paris, Strie D 250, 2439-2441.
Scientia Horticulturae 72 (1997) 59-62
Short communication
In vitro rooting improvement of globe artichoke (cv. Camus de Bretagne) by GA, J.M. Morzadec
*, A. Hourmant
Laboratoire de Physiologic des Syst&nes Intt?gr& Unicersite’ de Bretagne Occidentale, 6 ALjenue Victor le Gorgeu, B.P. 809, 29285 Brest Cedex, France
Accepted 25 July 1997
Abstract Microrosettes of globe artichoke (Cynara scolymus L.) were subcultured in a medium with naphthalene acetic acid and kinetin. For rooting, shoots were placed on an auxinic rooting medium (2.68 PM naphthalene acetic acid) supplemented with or without gibberellic acid (GA,). Treatments with 2.9 and 14.4 FM GA, resulted in 80.5 and 92.3% root formation, respectively, whereas 28.8 /IM GA, did not significantly change the rooting rate produced by auxin alone (50%). Moreover, the number of roots per shoot significantly increased for plants grown on rooting media containing 2.9 and 14.4 PM GA,. Stimulation by GA, of rhizogenesis was accompanied with a large increase of leaf growth. 0 1997 Elsevier Science B.V. Keywords: Cynara scolymus L.; In vitro rooting; Gibberellic acid (GA,)
1. Introduction
The
globe
propagated
artichoke
vegetatively
(Cynara by suckers
scolymus originating
L.) is a perennial from
axillary
buds
rosette
plant
on the basal
usually part of
et al., 1983; Basnizki and Goldschmidt, 1994). The efficiency of this method is however limited by the small proportion of axillary buds which produce viable cuttings (Benoit and Ducreux, 198 1). Besides, some heterogeneity is also introduced by the location of buds on mother plants producing differences in the the hypogeal
stem (Ryder
* Corresponding author. Tel.: +33 298 01 66 92; fax: +33 298 01 71 70; e-mail: jeammichel. [email protected] 0304.4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(97)00112-X
60
J.M. Morzadec, A. Houtmant/Scientia
Horticulturae 72 (1997) 59-62
inherent physiology of transplanted cuttings (Foury, 1976). In vitro vegetative multiplication techniques could, therefore, provide a way to correct these shortcomings. In vitro plant regeneration and propagation of globe artichoke has been studied for some forty years (Toponi, 1960). Microrosettes that develop have been found to be difficult to root, even in the presence of auxins. Depending on the cultivar, various results have been reported ranging from 1% rooted plants (Benoit and Ducreux, 1981) to 77% (Bigot and Foury, 1984) with 30% for cv. Camus de Bretagne (Moncousin, 1980). Because unrooted plants give rise to high mortalities during acclimatization, the present study was undertaken to improve rooting of cv. Camus de Bretagne. Gibberellins have been shown to inhibit rooting (Burkhart and Meyer, 1991); however, at low concentrations, they can stimulate it (Tizio et al., 1970). Gibberellins have long been known to play a major role in the organogenesis of rosette plants such as the globe artichoke (Basnizki and Goldschmidt, 1994). For these reasons, we have investigated the effects of the gibberellic acid (GA,) on rooting cv. Camus de Bretagne cultivated in vitro.
2. Materials
and methods
In vitro stock cultures of globe artichoke cv. Camus de Bretagne obtained from Organisation Bretonne de Selection (Plougoulm, France) were used as the source material. Cultures were subcultured for proliferation every 3 weeks on Murashige and Skoog (1962) basal medium supplemented with vitamins and Fe-EDTA (Linsmaier and Skoog, 1965), NaH,PO, (0.71 mM), adenine sulfate (0.22 mM), kinetin (2.3 PM), N6-(2-isopentenyl)adenine (2.46 PM) and naphtalene acetic acid (NAA) (0.54 PM) (PCcaut and Martin, 1993). For rooting, microrosette shoots selected presented at least a size of 5 cm and were cut 2 cm above their base before being transferred to a rooting medium. The Murashige and Skoog (1962) basal medium with l/2 strength for macroelements supplemented with vitamins and Fe-EDTA (Linsmaier and Skoog, 19651, 2 g 1-l charcoal activated, 2.68 PM NAA (medium A) was used and various concentrations of GA, 2.9, 14.4 and 28.8 PM were added. All media were supplemented with 30 g 1-l sucrose and 8 g 1-l or 7 g l- ’ agar type-E (Sigma Chemical, St. Louis, USA) for proliferation medium or rooting media, respectively. The pH of the complete media was adjusted to 5.8 with NaOH before autoclaving at 120°C for 20 min. Cultures were propagated in 15 X 2.2 cm tubes with 10 ml culture medium covered with clean plastic caps. Plants were grown at 23°C f 1, under 16 h day-’ light with an intensity of 100 PE me2 s-’ provided by Sylvania Grolux fluorescent lamps. Percentages of rooting were determined once a week during 6 weeks; the number of roots per shoot and analysis of fresh and dry weights were performed on rooted rosette shoots 6 weeks after subculturing. Results are the means of 4 independent experiments with 40 shoots per condition. Statistical analyses were performed using x2 tests and means separation using the Student’s t-test with a significance level of 5%.
J.M. Morzadec, A. Houmant/Scientia
Horticulturae
72 (1997) 59-62
61
3. Results and discussion Rosettes subcultured on rooting media (A, 2.9, 14.4 or 28.8 FM GA,) developed only one shoot apices per rosette. When globe artichoke rosettes raised in vitro on the multiplication medium were transferred to the rooting auxinic medium (A), a poor rooting of the plants was observed during the first three weeks; then the percentage of rooted explants started to increase, reaching 50% at week 6 (Fig. 1). The addition of GA, to the auxinic medium improved the rooting rate depending on GA, concentration. At the lowest concentrations (2.9 and 14.4 PM), GA, stimulated significantly (P = 0.05) the root formation after two weeks of culture. At 14.4 ,uM, GA, was significantly (P = 0.05) more effective than at 2.9 PM, producing at week 6 a high rooting percentage that was 92.3% (Fig. 1). The addition to the culture medium of a higher concentration of GA, (28.8 PM) did not change significantly (P = 0.05) the percentage of rooted explants compared to the auxinic medium (A); this was true through all the duration of culture (Fig. 1). The action of GA, on the root formation seems to be associated with an effect on the growth of microrosettes. Thus, at week 6, GA, (2.9 and 14.4 PM) significantly stimulated both the fresh and dry weights of rooted rosette shoots while 28.8 PM GA, did not have any effect (Table 1). The mean number of roots per shoot was statistically increased with 2.9 and 14.4 PM GA, whereas 28.8 PM GA, had no significant effect (Table 1). The data shows an unusual stimulating effect of GA, in rooting of a rosette plant; this phytohormone increased both the percentage of rooting (Fig. 1) and the number of roots per plant (Table 1). Because an earlier root appearance in GA,-treated shoots
Fig. 1. Effects of different concentrations
of GA, added to medium A on rooting of globe artichoke plants.
62
J.M. Morzadec, A. Hourmant/Scienria
Horticulturae 72 (1997) 59-62
Table 1 Effects of GA, added to the auxinic medium (A) on the fresh weight @WI, the dry weight (DW) of rooted rosette shoots and on the number of roots. Results expressed in g FW or DW ( f SE) and in number of roots per plant. Data recorded after 6 weeks of rooting induction. Means within a line followed by the same letter are not significantly different at P = 0.05 according to the Student’s t-test
Leaf FW Leaf DW Number of roots
Medium A
GA, (PM) 2.9
14.4
28.8
1.84*0. 13 a 0.14&O. 01 a 1.85+0. 15 a
2.71 +O. 17 b 0.19*0. 01 b 2.25&O. 13 b
3.41 +o. 15 c 0.23+0. 01 c 2.30*0. 09 b
1.79+0. 14 a 0.13*0. 01 a 1.95&O. 12 a
could facilitate the absorption of both nutrients and water, one could explain the improved growth of leaves recorded after 6 weeks of culture (Table 1). The use of GA, in the rooting medium conducts to a rapid root expression and to explants of high quality. Additional studies could be realised with other cultivars of globe artichoke especially cv. Violet de Provence known to be difficult to root.
References Basnizki, .I., Goldschmidt, E.E., 1994. Further examination of gibberellin A, effects on flowering of globe artichokes (Cynara scolymus L.) under controlled environment and field conditions. Isr. J. Plant Sci. 42, 159-166. Benoit, H., Ducreux, G., 1981. Etude de quelques aspects de la multiplication vCg&ative in vitro de l’artichaut (Cynara scolymus L.). Agronomie 1, 225-230. Bigot, C., Foury, C., 1984. Multiplication in vitro d’artichaut (Cynara scolymus L.) ZI partir de semences: Comparaison au champ de quelques clones ?I la lignte dont ils sont issus. Agronomie 4, 699-710. Burkhart, L.F., Meyer, M.M. Jr., 1991. The gibberellin synthesis inhibitors, ancymidol and flurprimidol, promote in vitro rooting of white pine microshoots. Plant Cell Rep. 10, 475-476. Foury, C., 1976. L’Artichaut Bul. Tech. Informa. 311, 415-432. Linsmaier, E.M., Skoog, F., 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18, 100-127. Moncousin, C., 1980. Multiplication vtgCtative acc&rie de Cynara scolymus L. I. RCsultats prkliminaires. Rev. Hart. Suisse 53, 149-154. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497. PBcaut, P., Martin, F., 1993. Variation occurring after natural and in vitro multiplication of early Mediterranean cultivars of globe artichoke (Cynara scolymus L.). Agronomie 13, 909-919. Ryder, E.J., de Vos, N.E., Bari, M.A., 1983. The globe artichoke (Cynara scolymus L.). HortScience 18, 646-653. Tizio, R., Saussay, R., Gautheret, R., 1970. Action de quelques gibtirellines sur la rhizogenese de fragments de rhizomes de Topinambour cultivts in vitro. C. R. Acad. Sci. Paris, S&e D 270, 2088-2092. Toponi, M., 1960. Recherches sur le d&eloppement de fragments de bracdes d’artichaut. C. R. Acad. Sci. Paris, Strie D 250, 2439-2441.